The ability to identify and isolate nucleic acid sequences which are unique to one of two cellular or body-fluid sources has important applications in medicine. One application is in understanding and predicting certain disease states, based on the presence or absence of a given messenger RNA (mRNA) species. For example, an absent or altered mRNA coding for a specific protein in a particular cell type is often the direct cause of a hereditary disease, while the presence of an added mRNA species may signal the beginning of malignant transformation or the latent presence of an otherwise undetectable infectious agent. Although some hereditary diseases--such as sickle cell anemia, other hemoglobinopathies and the thalassemias--are due to changes in the nature or presence of high-abundance mRNAs, a large percentage of hereditary diseases have been shown to be or are likely to be caused by the absence of or alterations in specific proteins coded for by low-abundance mRNAs. These include Lesch-Nyhan Syndrome, Hunter's Syndrome, Hurler's Syndrome, Tay-Sachs Disease and adenosine deaminase deficiency, among others (Stanbury).
It is also known that several oncogenes exist whose aberrant activation leads to malignant transformation (Van Beverow), and the detection of changes in mRNA species will have important applications in the early detection of such transformation.
Another application of unique-sequence isolation methods is in the diagnosis and study of viral or other cell or fluid borne agents. For example, the transcription of virus-specific mRNA(s) may be the first indication of reactivation of a latent viral infection (Kauffman). The method may also be used to isolate and identify viral agents.
Methods of isolating unique genomic sequences have applications to the study of gene expression during cell activation, embryonic development or cell cycle progression, and for analyzing genetic diseases which are related to the presence or absence of disease-related genomic regions.
One major problem in the detection and isolation of unique mRNAs, complementary DNAs (cDNAs), or genomic fragments, is interference caused by numerous high-abundance species present in the source material. For example, in any given cell type, there may be 10,000-30,000 distinct mRNA species (Davidson), and these can range in concentration from several hundred thousand molecules per cell, for high-abundance species, to only a few molecules per cell, for low-abundance species. Thus, if the unique species of interest has a relatively low-abundance, the ratio of unique species may be as low as 1 in 10.sup.6.
A number of nucleic acid hybridization techniques aimed at isolating and analyzing unique nucleic acid species have been developed heretofore. These techniques rely on the ability of denatured nucleic acids to reassociate in a sequence specific manner by Watson-Crick base pairing interactions (Britten 1968, 1985). The rate at which renaturation occurs is determined by the sequence complexity of the sample, the absolute and relative concentrations of various species, the length of DNA fragments, and the conditions under which the reaction takes place (Hames).
In one hybridization method, a selected gene probe cDNA is labeled and added in single-strand form to a saturating mixture of mRNA transcripts. The presence of the probe-related transcript species can be assessed by the amount of labeled probe cDNA incorporated into double-strand material. This method is generally suitable for high- and moderate-abundance transcripts, but lacks the sensitivity required for the identification and analysis of low-abundance mRNAs due to high background levels.
Filter hybridization to a conventional cDNA library may be used to identify differences in low-abundance mRNAs from two different sources (Anderson). However, the one million or more library clones needed to insure the presence of virtually all low-abundance mRNAs effectively eliminates the utility of this technique for screening programs.
Nucleic acid subtraction techniques have also been proposed for use in studying differences in low-abundance mRNAs between related cell types (Maniatis). Here mRNA or cDNA preparations from two different sources are hybridized to completion, and the unhybridized remainder is examined for the presence of species of interest. This method, as it has been practiced heretofore, requires relatively large amounts of mRNA and/or cDNA starting material, especially where the species of interest is a relatively low-abundance mRNA. This limitation precludes the use of the method for many body-fluid or cellular samples of interest where the total concentration of unique species is low.